Methods, pharmaceutical formulations and kits for identification of subjects at risk for cancer

ABSTRACT

Subjects at risk for developing cancer may be identified by obtaining samples of diagnostic cells from the subjects and determining a measure of cytotoxicity of the cells, the measure of cytotoxicity correlating negatively with the risk of developing cancer. The development of cancer may be prevented in subjects determined to be at risk for developing cancer by administering priming and activating agents to the subject, by increasing the expression of A 1  adenosine receptors in cells of the subject, and increasing the affinity of cells of the subject for A 1  adenosine receptor ligands. The preventative and diagnostic methods of the present invention may be carried out with kits and pharmaceutical liposomal formulations.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/569,394, filed May 12, 2000, which in turn claims thebenefit of U.S. Provisional Application No. 60/134,276, filed May 14,1999, which applications are incorporated herewith in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods, pharmaceuticalcompositions and kits useful in identifying subjects at risk fordeveloping cancer, and additionally relates to methods, pharmaceuticalcompositions and kits useful in preventing the development of cancer inat-risk individuals.

BACKGROUND OF THE INVENTION

[0003] Circulating monocytes derived from bone marrow promonocytesmigrate into tissues where they differentiate into mature macrophages.Macrophages are a major component of the lymphoreticular infiltrate oftumors. Tumor-associated macrophages can be activated withimmunomodulators and other biological agents to kill tumor cells.Andreesen, Res. Immunol. 144:291 (1993). The tumoricidal effect ofmacrophages is selective for tumor cells, is independent oftumor-specific antigens or transplantation antigens, and is dependent ondirect cell-to-cell contact and the macrophage-to-tumor cell ratio.Whitworth, et al., Cancer and Metastasis Rev. 8:319 (1989). While thetumoricidal effect of macrophages appears to be unrelated to theresistance of the tumor cell to chemotherapy, it is also thought thatunder certain circumstances, tumor burden may exceed the destructivecapability of available activated macrophages. Thus, the efficacy ofactivated macrophages to destroy tumor cells may be dependent on (1) thepresence of a sufficient number of functioning, activated tumoricidalmacrophages at the tumor site, and (2) recruitment of circulatingmonocytes which, following differentiation to macrophages, bind anddestroy tumor cells. However, tumor-associated macrophages from mouseand human malignant tumors and their metastases have not been previouslyshown to be potent effectors of tumoricidal activity in the absence ofstimulation. See Mantovani, Immunol. Today 13:265 (1992).

[0004] Several approaches to cancer treatment with activated monocytesand macrophages have been reported. Some of these approaches are basedon the recognition that activated human blood monocytes andmonocyte-derived macrophages possess cytotoxic effects against tumorcell lines when stimulated by various biological agents, including gammainterferon (IFN-γ), lipopolysaccharide (LPS), or granulocyte macrophagecolony stimulating factor (GM-CSF). These approaches include activationof macrophages by local or systemic administration of bacterial products(including LPS), activation of circulating monocytes with intravenousliposomes containing immunomodulators (e.g.,N-acetylmuramyl-L-analyl-D-isoglutamyl-L-analyl-2-(1′,2′-dipalmitoyl)-sn-glycerol-3′-phosphorylethylamide(MTP-PE)), and local or intravenous “adoptive cellular immunotherapy.”See, e.g., Whitworth, et al., Cancer and Metastasis Rev. 8:319 (1989);Salgaller and Lodge, J. Surg. Oncol. 68:122 (1998); Hennemann, et al.,Cancer Immunol. Immunother. 45:250 (1998); Andreesen, et al., CancerDetect. Prev 15:413 (1991); Eymard, et al, Eur. J. Cancer 32A: 1905(1996); Faradji, et al., Cancer Immunol. Immunother. 33:319 (1991);Andreesen, et al., Cancer Res. 50:7450 (1990); Otto, et al., Eur. J.Cancer 32A: 1712 (1996). For example, methods of adoptive cellularimmunotherapy with differentiated, tumor-cytotoxic and invitro-generated macrophages have been developed. These methods weredeveloped in order to overcome local factors affecting maturation orsuppression of functional activation of tumor site-restrictedmacrophages. Following leukapharesis of a patient afflicted with cancer,autologous cells are reinfused into the patient with increasing dosesand over several weeks. This method has been attempted both with andwithout prior treatment of the patient with intravenous immunomodulators(e.g., GM-CSF) to increase the yield of monocytes and macrophages. Priorto reinfusion into the patient, differentiation of the monocytes tomacrophages is accomplished in vitro, followed by in vitro activation ofthe differentiated macrophages with immunomodulators or other biologicalagents, such as IFN-γ or LPS. In some cases, these autologous,differentiated, and activated tumor-cytotoxic macrophages aresite-directed to the tumor and administered locally (e.g.,intraperitoneally). Andreesen and Hennemann, Pathobiology 59:259 (1991).However, clinical responses to the treatment of human malignancies, with(1) intravenous or local administration of activated autologousmacrophages; (2) intravenous liposomes containing immunomodulatingagents; or (3) local or systemic treatment with immunomodulators orbacterial products have not been reported.

[0005] In subjects suffering from malignant cancer, the cytotoxic effectof the subjects' monocyte/macrophage cells is impaired (i.e., thesecells lack spontaneous cytotoxic effects toward tumor cells). In onereport, less than 25% of cancer patients possessed macrophages capableof killing their own tumor cells. Cameron, et al., Cancer 53:2053(1984). It has also been shown that despite activation in vitro withLPS, monocyte/macrophage cells in patients with cancer lack cytotoxiceffects toward both autologous and allogeneic tumor cells. See Cameronand O'Brien, Cancer 50:498 (1982); Cameron, et al., Cancer 53:2053(1984); Key, Cancer Metastasis Rev. 2:75 (1983); Triozzi, et al., BoneMarrow Transplant. 18:47 (1996). This impairment may be a function ofmaturation or differentiation and/or responsiveness and function ofthese cells.

[0006] It has also been reported that following terminaldifferentiation, macrophages have a greater tumor cytotoxic effect thanmonocytes following stimulation with IFN-γ. Andreesen, et al., CancerRes. 50:7450 (1990). In one particular study, peripheral blood monocytesobtained from normal donors or breast cancer patients were testedimmediately after harvesting and activation; enhanced cytotoxicity wasnot observed. When monocytes were allowed to mature in vitro for 5 days,however, normal macrophages developed enhanced cytotoxicity while breastcancer patients' macrophages did not acquire enhanced cytotoxicity.Cameron and O'Brien, Cancer 50:498 (1982).

[0007] Cameron and O'Brien also reported that macrophages derived frommore than 50% of patients with colon and hematological malignancies werecytotoxic for allogeneic tumor cells derived from a colon cell line. Id.In this report, cytotoxicity was measured in terms of the percentage oftumor cells killed by activated macrophages, and a cytotoxicity ofgreater than 10% was considered statistically significant. The percentcytotoxicity of activated macrophages from patients with colon cancerfor allogeneic tumor cells ranged from 11 to 21%. Activated macrophagesobtained from patients with breast and gynecological tumors were notcytotoxic for tumor cells derived from allogeneic breast and prostatecell lines. Moreover, a macrophage inhibitory factor was demonstrated inthe plasma of patients with colon cancer with cytotoxic macrophages andin the plasma of patients with gynecological cancer with cytotoxicmacrophages. Out of 66 patients studied with either breast, colon,gynecological or hematological cancers, 47 out of 50 patients'macrophages were incapable of killing allogeneic tumor cells in vitroeither because they were unresponsive to activation or because of thepresence of an inhibitory factor in their plasma. It was concluded thatalthough some patients with cancer may possess macrophages that can beactivated in vitro, they may be ineffective in vivo.

[0008] Triozzi, et al. report that in patients with metastatic cancers,peripheral blood stem cells were mobilized with GM-CSF. Bone MarrowTransplant. 18:47 (1996). Spontaneous monocyte/macrophage tumorcytotoxicity was not detectable, either before or aftercryopreservation. However, cell cytotoxicity was inducible in vitro withIFN-γ against K562 cells with an effective monocyte/macrophage to tumorcell ratio of 20-10 to 1. The tumoricidal effect of these cells againstthe patients' own tumor cells was not tested. In another report,spontaneous monocyte-mediated cytotoxicity (SMMC) was severely depressedin patients with malignancies as compared with normal controls [7%(malignancies) vs. 43% (normal)]. Kleinerman et al., Lancet ii:1102(1980). This same study showed that in seven patients with advancedovarian cancer, treatment with cisplatin increased the SMMC at leastthree-fold. Id. In a third study, monocyte/macrophage tests, includingmacrophage precursor tests, antibody-dependent cellular cytotoxicitytests, spontaneous cellular cytotoxicity tests, and measurements ofmonocyte lysozyme activity were performed on monocytes from patientswith breast, colon, head and neck, lung, and melanoma cancers andcontrols. Unger, et al., Cancer 51:669 (1983). Selected assays ofperipheral blood monocyte function were abnormal in certain types ofcancer patients and normal in others, and did not show consistentcorrelations with tumor type or stage. Id.

[0009] Various mouse and human tumors release factors that arechemotactic for the migration of monocytes. Monocyte chemotactic protein(MCP-1) is chemoattractive for monocytes but is inactive for lymphocytesand neutrophils. Montovani, et al., Immunol. Today 13: 265 (1992). MCP-1is the human homologue of JE, a gene originally identified in mousefibroblasts and a member of the superfamily of cytokines calledchemokines. It interacts with a receptor that is coupled to a pertussistoxin-sensitive G protein to induce a rapid increase in intracellularfree calcium. In addition to tumor cells, fibroblasts, smooth musclecells, endothelial cells, and mononuclear cells themselves can produceMCP-1. Gene transfer of human MCP-1 into a mouse melanoma cell line hasbeen reported. Bottazzi, et al., J. Immunol. 148:1280 (1992). Aftersuccessful transformation, a prolongation of doubling time of the tumorcell lines and an increase in tumor associated macrophages was observed.However, there was no reduction in tumorigenicity.

[0010] LPS has been shown to activate macrophages to secrete cytokines,(including tumor necrosis factor-α (TNF-α), and to cause them to becometumoricidal Chen, et al., Curr. Topics in Microbiol Immunol. 181:169(1992). The expression of mouse and human genies for cytokineexpression, including TNF-α and interleukin-1β, are promoted by LPSactivation of nuclear translocation of nuclear factor κB (NF-κB) inmacrophages. Sweet and Hume, J. Leukoc. Biol. 60:8 (1996). Followingpriming with priming agents (e.g., phorbol myristoyl acetate (PMA),IFN-γ, f-met-leu-phe (fMLP), GM-CSF, lymphokines, muramyl dipeptide(MDP), muramyl tripeptide (MTP), and MTP-PE), macrophages can be fullyactivated by LPS. Priming is associated with an enhancedmetabolic/oxidative burst and the release of various oxygen metabolites,including superoxide anion and H₂O₂. Hamilton and Adams, Immunol. Today8:151 (1987). Moreover, priming induces macrophages to bind to tumorcells but, in the absence of activation, does not induce these cells tobe fully cytotoxic for tumor cells. Adams and Hamilton, Ann. Rev.Immunol 2:283 (1984).

[0011] Endotoxin (i.e., LPS) binds to and activates A₁ adenosinereceptors. Neely, et al., Am. J. Physiol. (Lung Cell Mol. Physiol.)272:L353 (1997). A₁ adenosine receptors are coupled to a pertussistoxin-sensitive G protein, and activation of this receptor increasesintracellular free calcium. Arend, et al., Am. J. Physiol. (CellPhysiol.) 255:C581 (1988). Moreover, human monocytes express A₁adenosine receptors and oxidative stress increases A₁ adenosine receptorexpression by NF-κB. Salmon, et al., J. Immunol. 151:2775 (1993); Nie,et al., Mol Pharmacol 53:663 (1998). It was recently reported thatadenosine inhibits tumor cell growth of the tumor cell lines Nb2lymphoma, K-562 leukemia, and LNCaP prostate carcinoma cells, whilestimulating proliferation of normal murine bone marrow cells. Fishman,et al., Cancer Res. 58:3181 (1998).

[0012] In 1974, a strain of mice that is resistant to LPS was developed.Watson and Riblet, J. Exp. Med. 140:1147 (1974). Following treatmentwith intravenous LPS, macrophages from LPS-sensitive C3H/HeN mice werefound to be tumoricidal. However, macrophages from LPS-resistant C3H/HeJmice could not be activated for tumor cytotoxicity. Ruco and Meltzer, J.Immunol. 120:329 (1978). C3H/HEJ mice have a genetic defect thatsignificantly reduces their ability to respond to LPS. Kraatz, et al.,Shock 11:58 (1999). This hyporesponsiveness to LPS has been confirmedboth in in vitro and in vivo studies. The precise nature of thismolecular defect in these mice is unknown. The ability of thesemacrophages to respond to LPS is controlled by a single gene, Lps^(d),which is located on chromosome 4. This defect is restricted to theinability of C3H/HeJ mice to respond to LPS; all other known functionsof cells from these mice are normal. Some investigators believe thisdefect is related to an early signal transduction event and the abilityto produce cytokines, such as TNF-α. However, the precise signaltransduction defect is not known. Ruco and Meltzer, J. Immunol. 120:329(1978); Sakagami, et al., Infect. Immun. 65:3310 (1997). Macrophageselicited from C3H/HeJ mice by Bacillus Calmette-Guerin (BCG) bind totumor cells. However, only in C3H/HeN macrophages did LPS induce theexpression of TNF-α. Adams and Hamilton, Ann. Rev. Immunol 2:283 (1984);Sweet and Hume, J. Leukoc. Biol. 60:8 (1996).

[0013] In spite of the foregoing, it has heretofore not been appreciatedthat a predictive relationship exists between the cytotoxicity of asubject's macrophages and monocytes against cancer cells, and the riskof the development of cancer in the subject. The tumoricidal effects ofprimed and activated macrophages and monocytes have not been examined inthe context of developing diagnostic methods for determining a subject'srisk of developing cancer.

SUMMARY OF THE INVENTION

[0014] The present invention is based on the inventor's realization thata reliable predictor of a subject's likelihood or susceptibility todeveloping cancer is the measure of cytotoxicity of certain diagnosticcells (i.e., macrophages or monocytes) of the subject for target cancercells. The measure of cytotoxicity may be an indirect measure, such as ameasure of the number of A₁ adenosine receptors (A₁AR) produced by thediagnostic cells, or a measure of the affinity of the diagnostic cellsfor A₁ adenosine receptor specific ligands (i.e., a measure of theaffinity of the A₁ARs present in the membranes of the diagnostic cellsfor A₁AR specific ligands), or a measure of the ability of the cells(i.e., the A₁ARs present in the membranes of the cells) to bind MCP-1protein, or a measure of the ability of the cells (i.e., the A₁ARspresent in the membranes of the cells) to bind annexins. Alternatively,the measure of cytotoxicity may be a functional measure of thecytotoxicity of the cell for cancer cells (i.e., a measure of theability and efficiency of the A₁ adenosine receptors of the cell tocouple to G proteins and signal-transduction-related pathways includingprotein kinase C, tyrosine kinase, phospholipase C, phospholipase D,phospholipase A₂, or NF-κB; a measure of the ability of the cell toinduce the release of cytotoxic substances or cytokines, includinginterleukin-1, TNF-α, or thromboxane; or a measure of the ability of thecells to kill target cancer cells). The measure of cytotoxicity isnegatively correlated to the risk of developing cancer; that is, thehigher the cytotoxicity of the subject's own cells for cancer cells, thelower the risk of the subject developing cancer. The inventor hasadditionally discovered that methods of increasing the cytotoxicity ofthe diagnostic cells in those subjects found to be at-risk fordeveloping cancer may actually prevent the development of cancer inthose subjects. Moreover, the inventor has discovered that cloned orcommercially available monocytes, macrophages, and precursors thereofthat have increased cytotoxicity against tumor cells may be used toprevent the development of cancer in at-risk patients.

[0015] One particular advantage of the present invention relates to theunfortunate fact that by the time most cancers are detectable by currentdetection methods, they are large or widespread enough to have alreadycaused significant morbidity in the subject. Accordingly, one advantageof the presently described diagnostic method for determining the risk ofdeveloping cancer is that preventative steps may be taken prior to theonset of disease. An advantage of the methods of preventing cancerdisclosed herein is that the onset, spread, or recurrence of cancer maybe partially or even completely prevented prior to the development of acancer that is detectable (i.e., when the cancer does not yet exist oris not detectable by current detection methods, or when the cancer is ina microscopic, micrometastatic or microvascular state), or after apreviously-existing cancer has been eradicated or “cured” by priortreatments.

[0016] Accordingly, a first aspect of the present invention is a methodof determining a subject's risk for developing cancer, carried out byobtaining a sample of diagnostic cells (e.g., macrophages, monocytes,promonocytes, peripheral blood stem cells) from a subject; and thendetermining a measure of cytotoxicity of the diagnostic cells for targetcancer cells, the measure of cytotoxicity correlating negatively withthe risk for developing cancer. The measure of cytotoxicity may be anindirect measure of cytotoxicity or a functional measure ofcytotoxicity.

[0017] A second aspect of the present invention is a method ofpreventing cancer in a subject at risk for developing cancer, carriedout by administering to the subject a priming agent in an amounteffective to prime macrophages of the subject. Optionally, an activatingagent may also be administered to the subject.

[0018] A third aspect of the invention is a method of preventing cancerin a subject at risk for developing cancer, carried out by increasingthe expression of A₁ adenosine receptors in the cells of the subject,the cells being selected from the group consisting of monocytes,macrophages, promonocytes and peripheral blood stem cells.

[0019] A fourth aspect of the invention is a method of preventing cancerin a subject at risk for developing cancer, carried out by increasingthe affinity of the cells of the subject for A₁ adenosine receptorligands, the cells being selected from the group consisting ofmonocytes, macrophages, promonocytes and peripheral blood stem cells.

[0020] A fifth aspect of the invention is a pharmaceutical liposomalformulation for the prevention of cancer in a subject determined to beat risk for developing cancer, comprising a priming agent and anactivating agent encased in liposomes.

[0021] A sixth aspect of the invention is a diagnostic kit fordetermining a subject's risk for developing cancer comprising at leastone reagent for determining the cytotoxicity of diagnostic cells of thesubject, and printed instructions for assessing the subject's risk fordeveloping cancer, packaged together in a container.

[0022] A seventh aspect of the invention is a kit for preventing cancerin a subject determined to be at-risk for the development of cancer,comprising at least one reagent selected from the group consisting ofreagents for increasing A₁ adenosine receptor expression in macrophages,monocytes, promonocytes and peripheral blood stem cells, reagents forincreasing binding of A₁ adenosine receptor ligands to macrophages,monocytes, promonocytes and peripheral blood stem cells, reagents forincreasing binding of MCP-1 protein for macrophages, monocytes,promonocytes and peripheral blood stem cells, priming agents andactivating agents; and printed instructions for administering the atleast one reagent to the subject, packaged together in a container.

[0023] The foregoing and other aspects of the present invention areexplained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a graphical illustration showing that following primingwith PMA (1 μm) and activation with 2-chloro-N⁶-cyclopentyladenosine(CCPA) (lam), macrophages from LPS-sensitive mice (IC-21, ATCC) produce50% cytotoxicity of mouse B-16 melanoma cells versus 20% cytotoxicityproduced by macrophages from LPS-resistant mice (WBC264-9C, ATCC).

[0025]FIG. 2 is a graphical illustration showing that following primingwith PMA (1 μM) and activation with an A₁ AR agonist, CCPA (1 μM), mousemacrophages (IC-21, ATCC) are cytotoxic for mouse P815 mastocytoma tumorcells.

[0026]FIG. 3 is a graphical illustration showing that following primingwith PMA (1 μm) and activation with CCPA (1 μm), human macrophages (SChematopoietic cell line, ATCC and a healthy volunteer) are cytotoxic toA375 human melanoma tumor cells.

[0027]FIG. 4 is a graphical illustration showing that in saturationexperiments, A₁ adenosine receptors are expressed in macrophages fromboth LPS-sensitive mice (IC-21, ATCC) and LPS-resistant mice (WBC264-9C,ATCC) [B_(max) 420 (LPS-sensitive) versus B_(max) 425 (LPS-resistant)];however, the affinity (K_(d)) of A₁ adenosine receptors for a highlyselective A₁ adenosine receptor ligand, ¹²⁵ I BWA844U, is greater inLPS-sensitive mouse macrophages than LPS-resistant mouse macrophages[K_(d)=11.83 (LPS-sensitive) macrophages versus K_(d)=22.47(LPS-resistant) macrophages].

[0028]FIG. 5 is a graphical illustration showing that in saturationexperiments, treatment of human macrophages (SC, ATCC) with cisplatin(2.5 μM) for 24 hours increased A₁ adenosine receptor expression byapproximately three-fold.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a generalized strategy foridentifying subjects at risk for developing cancer, and for preventingcancers in subjects identified as being at risk for developing cancer.An at-risk subject is any individual who is believed to be at a higherrisk than the general population for developing cancer.

[0030] The present invention is suitable for both medical and veterinaryuses. Suitable subjects include, but are not limited to, mammalian andavian subjects. More preferred subjects are mammalian subjects such ashumans, monkeys, pigs, cattle, dogs, horses, cats, sheep, and goats. Themost preferred subjects are human subjects.

[0031] The term “cancer” as used herein is intended to encompass cancersof any origin, including both tumor-forming and non-tumor formingcancers. The term “cancer” has its understood meaning in the art, forexample, an uncontrolled growth of tissue that has the potential tospread to distant sites of the body (i.e., metastasize). As used herein,the term “cancer cell” is also intended to encompass those cellsreferred to as “precancerous,” i.e., cells that contain mutated ordamaged DNA or other components, which mutations or damage are likely tocause the cell to develop into a cancer cell. Exemplary cancers includeosteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas;leukemias; sinus tumors; ovarian, uretal, bladder, prostate and othergenitourinary cancers; colon, esophageal and stomach cancers and othergastrointestinal cancers; lung cancers; lymphomas; myelomas; pancreaticcancers; liver cancers; breast cancers; kidney cancers; endocrinecancers; skin cancers; melanomas; angiomas; and brain or central nervoussystem (CNS) cancers. Tumors or cancers, as defined herein, may be anytumor or cancer, primary or secondary, which is recognized by cytotoxiccells (for example, macrophages) and which induces the tumoricidaleffect of the cells upon contact. See, e.g., Alexander and Evans, NatureNew Biology 232:76 (1971). Preferred are methods of identifying subjectsat risk for tumor-forming cancers, and methods of preventing the same.The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multi-cellularorganism. Tumors can be malignant or benign. Preferably, the inventivemethods disclosed herein are used to identify subjects at risk fordeveloping malignant tumors, and to prevent malignant tumors.

[0032] The methods of the present invention are also useful indetermining the risk of a subject developing a non-cancer disorder ofcell proliferation. These disorders include, but are not limited tohyperplasias, hyperpigmentation of the skin, psoriasis, lipomas,fibroids, keloids, adhesions, and any other disorder wherein cellproliferation is uncontrolled.

[0033] These methods are based upon the inventive premise that themeasure of cytotoxicity of certain cells (herein described as“diagnostic cells”) of the subject for target cancer cells is a reliablepredictor of the risk of developing cancer. For example, humanmonocytes/macrophages can be tested by methods of the present inventionto determine a measure of the cytotoxicity of these cells for humantumor cell lines. The measure of cytotoxicity is negatively correlatedto risk for developing cancer; that is, a subject whose diagnostic cellsexhibit a high cytotoxicity for cancer cells is at a lower risk for thedevelopment of cancer than a subject whose diagnostic cells exhibit alower cytotoxicity for cancer cells.

[0034] In the inventive method of the present invention, a sample ofdiagnostic cells is first obtained from the subject. Obtaining a sampleof diagnostic cells from the subject may be carried out by any methodknown in the art (e.g., isolating cells from blood samples). Diagnosticcells, as used herein, are cells that express A₁ adenosine receptors andinclude, but are not limited to, monocytes, macrophages, promonocytes,peripheral blood stem cells (PBSC), and hematopoietic stem cells withmonocytes and macrophages being preferred.

[0035] After being obtained from the subject, the diagnostic cells arethen tested for cytotoxicity for target cancer cells, the results of thetesting providing a measure of cytotoxicity of the subject's diagnosticcells. The measure of cytotoxicity may be an indirect measure ofcytotoxicity. Examples of indirect measures of cytotoxicity include ameasure of the number of A₁ adenosine receptors (A₁AR) in the membranesof the diagnostic cells, or a measure of the affinity of the diagnosticcells for A₁ adenosine receptor specific ligands (i.e., a measure of theaffinity of the A₁ARs present in the membranes of the diagnostic cellsfor A₁AR specific ligands), or a measure of the ability of thediagnostic cells (i.e., the A₁ARs present in the membranes of the cells)to bind MCP-1 protein, or a measure of the ability of the diagnosticcells (i.e., the A₁ARs present in the membranes of the cells) to bindannexins. The measure of cytotoxicity may also be a functional measureof cytotoxicity as defined herein, in which case the diagnostic cellsare primed and activated prior to being tested for cytotoxicity.

[0036] One indirect method of determining the measure of cytotoxicity ofdiagnostic cells for target cancer cells is evaluating the number of A₁adenosine receptors in the membranes of the diagnostic cells accordingto methods known in the art (e.g., by determining B_(max) using labeledligand saturation binding techniques, where B_(max) is an expression ofthe density or number of A₁ adenosine receptors present in the membranesof the cells). In general, the higher the number and/or density of A₁adenosine receptors on the diagnostic cells, the greater the measure ofcytotoxicity of the diagnostic cells for target cancer cells, and thusthe lower the risk of the subject for developing cancer.

[0037] A second and more preferred indirect method of determining ameasure of cytotoxicity of the diagnostic cells of the subject isevaluating the affinity of the diagnostic cells for A₁ adenosinereceptor (AR) ligands. Alternatively, the affinity of the diagnosticcells for MCP-1 protein, or the affinity of the diagnostic cells forannexins, may be evaluated. These measurements of affinity may becarried out using labeled ligand binding measurement techniques known inthe art. Ligands may be labeled with radioactive compounds, fluorescentcompounds, biotinylated compounds, and the like. These methods ofevaluating the affinity of the diagnostic cells include using saturationbinding techniques or competitive binding techniques to determine theaffinity of the diagnostic cells for A₁AR ligands, or for MCP-1 protein,or for annexins, as expressed by K_(d) (saturation binding experiments)or K_(i) (competition binding experiments), with the value of K_(i) andK_(d) being inversely related to affinity (i.e, the lower the K_(i) orK_(d), the higher the affinity). In general, the lower the value of theK_(d) or K_(i) of the diagnostic cell, the greater the measure ofcytotoxicity of the diagnostic cell, and the lower the risk of thesubject developing cancer.

[0038] Alternatively, or additionally, a measure of cytotoxicity of thediagnostic cells may be a functional measure of cytotoxicity. Afunctional measure of cytotoxicity refers to a measure of at least oneindicia of tumoricidal activity (e.g., release of cytotoxins orcytokines by the cells, or the percentage of killed tumor cells by thediagnostic cells) exhibited by the diagnostic cells. In the practice ofthe present invention, diagnostic cells are primed and activated priorto determining a functional measure of cytotoxicity.

[0039] Diagnostic cells are primed by contacting the cells with apriming agent in an amount sufficient to prime the diagnostic cell, andfor a time sufficient to prime the diagnostic cell. As used herein,“contacting” a cell with a substance means (a) providing the substanceto the environment of the cell (e.g., solution, in vitro culture medium,anatomic fluid or tissue) or (b) applying or providing the substancedirectly to the surface of the cell, in either case so that thesubstance comes in contact with the surface of the cell in a mannerallowing for biological interactions between the cell and the substance.

[0040] As used herein, “priming” of macrophages refers to a treatmentwhich enhances the metabolic burst of macrophages, wherein the metabolicburst (see Hamilton and Adams, Immunol. Today 8:151 (1987)) is increasedover that which would occur in the absence of priming. As used herein,“primed” macrophages refers to those that have undergone a primingtreatment; “primers” or “priming agents” refer to agents capable ofpriming macrophages. The diagnostic cells may be primed using anypriming agent known in the art, including but not limited to PMA (see,e.g., Leaver, FEMS Microbiol. Immunol. 47:293 (1989); White, J. Biol.Chem. 259:8605 (1984)); lipopolysaccharide (LPS) (see, e.g., Glaser, J.Biol. Chem 265:8659 (1990); Pace, J. Immunol. 126:1863 (1981);Alexander, Nature New Biol. 232:76 (1971)); platelet activating factor(PAF) (see, e.g., Stewart, Immunology 78:152 (1993); Salzer, J. Clin.Invest. 85:1135 (1990)); tumor necrosis factor alpha (TNF_(α)) orthrombin (see, e.g., Stewart, Immunology 78:152 (1993)); f-met-leu-phe(FMLP) (see e.g., Stewart, Immunology 78:152 (1993)); zymosan (Rankin,J. Clin. Invest. 86:1556 (1990); macrophage stimulating factorsincluding granulocyte macrophage colony stimulating factor (GM-CSF);ionomycin (for example in 1 μM amounts); calcium ionophore (such as A23187, for example in 0.1-10 μM amounts); gamma interferon (IFNτ, forexample in 1-150 units/ml amounts) Flebbe, J. Immunol. 145:1505 (1990);supernatants of tumor cells (Hamilton and Adams, Immunology Today 8:151(1987); Marvin, J. Surg. Res. 63:248 (1996)); heparin (Heinzelmann etal., Ann. Surg. 229:542 (1999), enoxaparin (Id.), or bacterial productsfrom gram positive organisms (see, e.g., Bacterial EndotoxinLipopolysaccharides, Morrison and Ryan (Eds.) CRC Press, Boca Raton,Fla., 1992; Hamilton and Adams, Immunology Today 8:151 (1987); Loppnow,Methods Enzymol. 236:3 (1994)). Preferred priming conditions for thetype of cell to be to be primed may be determined using routine methodsknown to those in the art.

[0041] After being primed, diagnostic cells are activated by contactingthe cells with an activating agent in an amount sufficient to activatethe diagnostic cell, and for a time sufficient to activate thediagnostic cell. As used herein, “activated” cells are those whichpossess tumoricidal functions. Suitable activating agents include andare preferably A₁ adenosine receptor agonists, including but not limitedto adenosine; cyclohexyladenosine; various N⁶-substituted A₁ adenosineagonists including but not limited to N⁶ cyclopentyladenosine, N⁶R-phenylisopropyladenosine, 2-chloro N⁶ cyclopentyl adenosine (CCPA), N⁶(p-sulfophenyl) alkyl and N⁶ sulfoalkyl derivatives of adenosine (suchas N⁶-(p-sulfophenyl) adenosine; 1-deaza analogues of adenosineincluding but not limited to N⁶ cyclopentyl 1-2-chloro-1-deaza adenosine(1-deaza-2-Cl-CPA); N cycloaklyladenosines; N⁶ bicycloalkyladenosines;ribose modified adenosine receptor analogues including but not limitedto 3′-deoxy-R-PIA. See, e.g., Conti, Naunyn-Schmiedeberg's Arch.Pharmacol. 348:108 (1993); Trivedi, J. Med. Chem. 32:8 (1989); Jacobsen,J. Med. Chem. 35:4143 (1992); Thedford, Expl. Cell. Biol. 57:53 (1989);Trewyn, Exp. Pharmacol. 28:607 (1979); Fleysher, J. Amer. Chem. Soc.(August 1968); Fleysher, J. Amer. Chem. Soc. November 1969));cycloalkyladenosines (see e.g., Moos, J. Med. Chem. 28:1383 (1985));analogs of R-PIA, CHA, and CPA (see, e.g., Cristalli, J. Med. Chem.31:1179 (1988)). Van der Wenden, J. Med. Chem. 38:4000 (1995); Jacobson,PJM Med. Res. Rev. 12:423 (1992); Daly, J. Med. Chem. 25:197 (1982).

[0042] Lipids may optionally be conjugated to the priming agent(s)and/or the activating agent(s) by techniques known in the art, in orderto increase the bioavailability and/or the affinity of the priming agentor activating agent for the cell.

[0043] After priming and activation, the diagnostic cells are thentested to determine a functional measure of cytotoxicity of thediagnostic cells for target cancer cells. As used herein, thetumoricidal or cytotoxic effects “of” diagnostic cells, or “by”diagnostic cells, or “for” target cancer cells refers to the presence ofcellular activities in diagnostic cells that allow the diagnostic cellsto have cytotoxic effects on target cancer cells. These effects can beassessed by analyzing the cellular components of the diagnostic cell(e.g., by evaluating an increase in enzymes or factors associated withsignal transduction pathways which produce cytotoxic compounds orcytokines), or by evaluating the cytotoxic compounds produced by thediagnostic cell, or by evaluating the tumoricidal effects of thediagnostic cell on an appropriate target cancer cell, as furtherdescribed herein. “Target cancer cells” include but are not limited tocells from known and described tumor cell lines. Alternatively, redblood cells may be used as target cells to test the cytotoxicity of thediagnostic cells. See Kleinerman et al., Lancet ii: 1102 (1980). Inpreferred embodiments of the invention, the target cells are of the samespecies as the subject. For example, if the subject is human, then thediagnostic cells are preferably tested for cytotoxicity against humantumor or cancer cells or human red blood cells.

[0044] A functional measure of cytotoxicity of the diagnostic cells maybe determined by several methods. In general, a functional measure ofcytotoxicity of the diagnostic cells is determined by testing thediagnostic cells using at least one of several alternate methods ofmeasuring the ability of the diagnostic cells to kill cancer or tumorcells, as indicated by at least one of several indicia. These methods oftesting are generally known in the art. For example, one method ofdetermining a functional measure of cytotoxicity useful in the presentinvention is by calculating or evaluating the percentage of tumor cellskilled by primed and activated diagnostic cells of the presentinvention. The tumor cells may be obtained from known tumor cell lines.In this method, the primed and activated diagnostic cells of the presentinvention are contacted with target cancer cells under conditions knownin the art. After contact with the primed and activated diagnosticcells, the percentage of tumor cells in the sample killed by the primedand activated diagnostic cells is calculated according to techniquesknown in the art. The percentage of tumor cells killed is then comparedto a standard or reference percentage of tumor cell killing in order todetermine if the subject is at risk for developing cancer. Such astandard percentage may differ from determination to determination basedon kind of tumor cell tested in the sample and other factors, and maychange with time based on the accumulation of additional data withregard to the statistical analysis of the development of cancer in apopulation, but will generally be known to the medical practitioner. Inone preferred embodiment of the invention, a percentage of tumor cellkilling by the primed and activated diagnostic cells of less than about10% indicates that the subject is at risk for developing cancer.

[0045] Another preferred method of determining a functional measure ofcytotoxicity is evaluating the concentration of A₁ adenosine receptorligand required to produce cytotoxic or tumoricidal functional effectsin the diagnostic cells, which functional effects include but are notlimited to the release of cytokines or cytotoxic substances (e.g.,TNF-α, interleukin-1, or thromboxane) by the diagnostic cells. Ingeneral, the lower the concentration of A₁AR ligand required to producea tumoricidal effect in the cell, the greater the measure ofcytotoxicity of the diagnostic cells, and the lower the risk of thesubject developing cancer. Alternatively, the amount of cytokinereleased by the primed and activated diagnostic cells can be measuredand correlated to risk of developing cancer. In general, the greater theamount of cytokine released by the diagnostic cells, the greater themeasure of cytotoxicity of the diagnostic cells, and the lower the riskof the subject developing cancer.

[0046] Other methods of determining a functional measure of cytotoxicityinclude, but are not limited to methods of evaluating the ability andefficiency of the A₁ adenosine receptors of the diagnostic cells tocouple to signal-transduction related pathways such as G-proteins,protein kinase C, phospholipase C, phospholipase D, phospholipase A₂, orNF-κB, according to techniques that are known to those skilled in theart.

[0047] As will be evident from the foregoing, the measure ofcytotoxicity of diagnostic cells may be determined, and preferably isdetermined by using more than one test for cytotoxicity. For example, amedical practitioner may determine a measure of cytotoxicity of thediagnostic cells of a subject both by calculating the percentage oftumor cells killed by the diagnostic cells (a functional measure ofcytotoxicity), and by determining the affinity of the diagnostic cellsfor A₁ AR ligands (an indirect measure of cytotoxicity). The combinationof results of these two tests thus provides the practitioner with morethan one indicia of whether or not the subject is at risk for developingcancer. Whether or not a subject is determined to be at risk fordeveloping cancer may be determined by the practitioner by comparing theresults of the tests for cytotoxicity with standards correlating ameasure of cytotoxicity with the risk of developing cancer.

[0048] The measure of cytotoxicity of the diagnostic cells positivelycorrelates with the likelihood of successful prevention of cancer in thesubject. Once a subject is identified as being “at risk” for developingcancer, inventive methods of the present invention may be used toprevent cancer in the subject. Although these methods may be applied tosubjects identified as being at risk by the methods of the presentinvention, subjects that are determined to be at risk for developingcancer as identified by other methods of identifying or assessing riskof cancer are also suitable subjects of the preventative and diagnosticmethods of the present invention. Additionally, “at risk” subjects mayalso be subjects that are defined as such because of other indicia ofcancer risk. For example, suitable subjects may include, but are notlimited to, individuals with a family history of cancer, individuals whohave previously been treated for cancer, individuals who have beenexposed to carcinogens (e.g., heavy smokers), individuals exposed tomedications or medical treatments associated with the development ofcancer (e.g., estrogens or radiation therapy), individuals determined tohave an increased likelihood of developing cancer by genetic testing,and individuals presenting any other clinical indicia suggesting thatthey have an increased likelihood of developing cancer. In particular,subjects who have been previously treated for cancer by known methods(e.g., radiation, chemotherapy) and who are deemed to be essentially“cancer-free” are suitable subjects for the preventative and diagnosticmethods of the present invention.

[0049] By the terms “prevention of cancer” or “preventing cancer” it isintended that the inventive methods eliminate or reduce the incidence oronset of cancer, as compared to that which would occur in the absence oftreatment. Alternatively stated, the present methods slow, delay,control, or decrease the likelihood or probability of cancer in thesubject, as compared to that which would occur in the absence oftreatment. It is specifically intended that the term “prevention ofcancer” includes the prevention of the recurrence of cancer in a subjectwho has previously been diagnosed with cancer and successfully treated.One therapeutic goal of the preventative methods of the presentinvention is to increase the cytotoxicity of the macrophages, monocytes,promonocytes and peripheral blood stem cells of the subject until thesecells are sufficiently cytotoxic for target cancer cells such thatcancer does not develop in the subject. In one embodiment of thepreventative methods of the invention, the cytotoxicity of themacrophages and/or monocytes of the subject is increased such that afunctional measurement of the percentage of tumor cells killed (as setforth in the description of diagnostic methods herein) by themacrophages and monocytes is at least about 30%.

[0050] The preventative methods of the present invention utilize thecytotoxic effects of primed and activated cells (including but notlimited to macrophages, monocytes, promonocytes and peripheral bloodstem cells) to prevent the growth of tumor cells. In essence, thepreventative methods of the present invention are useful in “arming” thecells of a subject at risk for cancer by increasing the cytotoxicity ofthe cells of the subject for target cancer cells. Preventative methodsof the present invention may be, and preferably are carried out in vivo.Increasing the cytotoxicity of the cells of the subject may be carriedout by one or more of several methods. For example, the present methodsmay be carried out by direct activation of the cells of the subject byadministering to the subject priming agents and/or A₁ adenosine receptoragonists (activating agents), either systemically or locally (i.e.,directly to the cells of the subject). In one embodiment of theinvention, a subject determined to be at risk for developing cancer issystemically administered at least one priming agent, as describedherein. The priming agent may be administered to the subject by any ofthe accepted methods of administering pharmaceutical compounds to asubject, including but not limited to administration parenterally (i.e,intravenously, subcutaneously, intradermally, intramuscularly,intraarticularly); orally, rectally, topically, (including buccal,sublingual, dermal and intraocular administration), transdermally and byinhalation. The dosage of the priming agent will be an amount effectiveto prime the cells (i.e., macrophages, monocytes, promonocytes,peripheral blood stem cells) of the subject and will vary with thecompound used and the condition or state (i.e., size, age) of thesubject. The daily dose may be divided among one or several unit doseadministrations. Treatments may continue on a chronic basis asnecessary. The priming agent being administered to the subject may beadministered to the subject by liposomal delivery according to methodsknown in the art, but may also be formulated into any acceptablepharmaceutical formulation (e.g., dissolved in a carrier suitable forinjection, admixed with a carrier in tablet or capsule form).

[0051] After administering a priming agent to the subject determined tobe at risk of developing cancer, the cytotoxicity of the diagnosticcells of the subject (i.e., macrophages and/or monocytes) may beevaluated according to the diagnostic methods of the invention set forthherein. Should the practitioner determine, using these diagnosticmethods, that a further increase in cytotoxicity of the subjectsmacrophages, monocytes, promonocytes or peripheral blood stem cells isdesired, then the present invention also provides for the administrationof an activating agent (i.e., an A₁ adenosine receptor agonist) inaddition to the administration of a priming agent. The activating agentmay be administered in conjunction with a priming agent of the presentinvention, or separately. The activating agent may be administered inessentially the same manner as the priming agent, as set forth herein.

[0052] In one embodiment of the invention, a priming agent and anactivating agent are formulated together (i.e., encapsulated in) in aliposomal formulation according to techniques known in the art, and thenadministered concurrently to the subject. See, e.g., U.S. Pat. Nos.5,527,528 to Allen et al.; U.S. Pat. No. 5,013,556 to Woodle et al.,U.S. Pat. No. 5,882,679 to Needham; and U.S. Pat. No. 5,766,627 toSamkaram et al, the disclosures of which are incorporated herein byreference. In a preferred embodiment of the invention, the liposomalformulation comprising the priming agent and the activating agent isformulated in a timed-release formulation such that the priming agent isreleased prior to the release of the activating agent.

[0053] Preventative methods of the present invention also includesubjecting certain cells (e.g., macrophages, monocytes, promonocytes,peripheral blood stem cells) of the subject with a number of differenttreatments to increase the expression of the human A₁ adenosine receptorand/or to increase the affinity of these receptors for A₁ adenosinereceptor ligands. These methods may be carried out in accordance withthe methods described in co-pending and co-owned U.S. patent applicationSer. No. 08/748,559, filed Nov. 8, 1996, the disclosure of which isincorporated herein in its entirety. Preventative methods that increasethe expression of human A₁ adenosine receptors in cells such as humanmonocytes, macrophages, promonocytes and peripheral blood stem cellsinclude: transfecting these cells with cDNA for the human A₁ adenosinereceptor gene; treating these cells with drugs, includingchemotherapeutic drugs, cisplatin, daunorubicin, doxorubicin, ormitoxantrone, dexamethasone, or other drugs such as carbamazepine;treating the cells with adenosine receptor antagonists (e.g.,theophylline); and/or subjecting the cells to ischemic conditions. SeeU.S. Pat. No. 5,320,962 to Stiles et al., Nie, et al., Mol. Pharmacol.53:663 (1998); Gerwins and Fredholm, Mol. Pharmacol. 40:149 (1991);Lupica et al. Synapse 9:95 (1991); Ren and Stiles, Mol. Pharmacol.55:309 (1999); Biber et al., Neuropsych. Pharmacol. 20:271 (1999).

[0054] Methods of preventing cancer in subjects deemed at risk fordeveloping cancer include methods that increase the affinity of A₁adenosine receptors (expressed in macrophages and monocytes, forexample) for A₁ adenosine receptor ligands. These methods of increasingthe affinity of A₁ adenosine receptors for A₁ adenosine receptor ligandsinclude contacting the cells expressing A₁ adenosine receptors withallosteric enhancers for A₁ adenosine receptors, such as PD 81,723 whichincreases the affinity and binding of an A₁ adenosine receptor ligandfor A₁ adenosine receptors and coupling of the receptor to the Gprotein; contacting the cells with divalent cations, including magnesiumand calcium; and/or contacting the cells with adenosine deaminase, orimmunomodulators or priming agents, such as lymphokines, MDP, MTP,MTP-PE, IFN-γ, PMA, GM-CSF, or FMLP. These treatments may be given invitro, in vivo, or in vivo, may be delivered by liposomes, and may beused in combination with treatments to increase A₁ adenosine receptorexpression as described herein.

[0055] Cells (e.g., macrophages, monocytes, promonocytes, peripheralblood stem cells) of the at-risk subject in whom prevention of cancer isdesired may also be treated with 1,25-dihydroxycholecalciferol toimprove terminal maturation of monocyte/macrophage cultures, and/orM-CSF or GM-CSF to stimulate the production of these cells. Bartholeyns,Res. Immunol. 144:288 (1993). These treatments may be used incombination with treatments to increase A₁ adenosine receptor expressionor binding of A₁ adenosine receptor ligands to these cells. Also,promonocytes obtained from the bone marrow or from peripheral bloodhematopoietic stem cells of the subject, or from commercial sources forhuman monocytes and human macrophages, may be tested for A₁ adenosinereceptor expression and affinity. These cells may also be transfectedwith cDNA for human A₁ AR gene, or treated with other drugs, includingchemotherapeutic drugs, dexamethasone, adenosine receptor antagonists,or subjected to ischemic conditions in order to increase A₁ ARexpression. These cells may also be treated with allosteric enhancersfor A₁ adenosine receptors, divalent cations, adenosine deaminase, orimmunomodulators or priming agents, as described above. See, e.g,Bhattacharya and Linder Biochimica and Biophys. Acta 1265:15 (1995);Musser et al., J. Pharmcol. Exp. Ther. 288:446 (1999). In addition,promonocytes, monocytes, peripheral blood stem cells, or macrophages ofthe subject may be transfected with cDNA for human MCP-1 protein orother cDNA for other human genes such as TNF-α or other immunomodulatoror priming agents, or adhesion molecules such as CD11/CD18, or ICAM-1,which promote hematopoietic growth, maturation, function, or survival incombination with cDNA for human A₁ adenosine receptor. These treatmentsmay induce these promonocytes, peripheral blood stem cells, monocytes,and macrophages to be more efficient and have greater cytotoxic effectstowards tumor cells and reduce the number of macrophages or themacrophage-to-tumor cell ratio necessary for an adequate tumoricidaleffect for these cells for the prevention of developing cancers in vivoby recognizing and destroying tumor cells and preventing tumor growth.

[0056] Finally, monocytes, macrophages, promonocytes and/or peripheralblood stem cells may be obtained from the subject or other sources(i.e., from other subjects, or from known cell lines, or from commercialsources), cloned to increase A₁ adenosine receptor expression, functionand tumoricidal effects, and then administered to the subject eithersystemically or locally. In a preferred embodiment of the invention,cells treated according to the methods described herein may beadministered to the subject in whom cancer prevention is desiredaccording to the administration techniques set forth in co-pending andco-owned application Ser. No 09/748,559, incorporated herewith in itsentirety.

[0057] Kits for determining a subject's risk of developing cancer willinclude at least one container sized to house at least one reagentuseful in determining a measure of cytotoxicity of diagnostic cells asdefined herein, and printed instructions for assessing whether or not asubject is at risk for developing cancer. Kits for preventing cancer ina subject will include at least one container sized to house at leastone reagent useful in preventing cancer according to methods describedherein, as well as printed instructions for carrying out these methods.As used herein, the term “reagent” means any compound, composition orbiological agent (i.e., samples, aliquots or “doses” of cells, cDNAs,recombinant DNAs, isolated genes, antibodies, etc.) useful in carryingout any method of the present invention, including but not limited topriming agents, activating agents, A₁ adenosine receptor ligands(including agonists, antagonists and antibodies to A₁ adenosinereceptors), antibodies and ligands for MCP-1 protein, antibodies andligands for annexins, antibodies and ligands for cytokines and cytotoxiccompounds produced by cells, cDNAs encoding A₁ adenosine receptors andcompounds useful in transfecting the cDNAs into cells, drugs and othercompounds for increasing A₁ adenosine receptor expression, drugs andother compounds for increasing affinity of cells for A₁ adenosinereceptors, drugs and other compounds for stimulating the production ofmonocytes and macrophages and/or the terminal differentiation thereof,buffers and carriers useful in isolating and preparing cells and/ormembranes for analysis and treatment, buffers and carriers useful incarrying out saturation and competition binding assays, allostericenhancers for A₁ adenosine receptors, known anti-cancer therapeuticcompounds, and radioactive and non-radioactive labeling compounds.

[0058] A diagnostic kit of the present invention may include reagentsfor performing indirect determinations of cytotoxicity of diagnosticcells (i.e., reagents for performing tests measuring the number of A₁adenosine receptors in the membranes of diagnostic cells, and/orreagents useful in performing tests measuring the affinity of diagnosticcells for A₁ adenosine receptor ligands and/or for MCP-1 protein).Accordingly, a diagnostic kit of the present invention may comprise acontainer containing at least one ligand for A₁ adenosine receptors,including antibodies for the A₁ adenosine receptor, A₁ adenosinereceptor agonists, and A₁ adenosine receptor antagonists. These reagentsmay be labeled with radioactivity or a non-radioactive label (i.e.,fluorescent label, biotinylated label), and are useful in measuring theK_(i) of these ligands for A₁ adenosine receptors in a competitionassay. A diagnostic kit may alternatively or additionally include acontainer comprising at least one ligand for MCP-1 or annexins,including antibodies for MCP-1 or annexins. These reagents are useful indetermining the K_(i) of these ligands for A₁ adenosine receptors in themembranes of a subject's diagnostic cells (e.g., in a competitionassay). Diagnostic kits for determining indirect measures ofcytotoxicity of diagnostic cells will include printed instructions forconducting the appropriate tests, and may include instructions forisolating diagnostic cells from a subject, instructions for isolatingand affixing to a solid support membranes from the diagnostic cells foruse in the tests, and instructions for conducting the tests themselves.The printed instructions will also include instructions for correlatingthe results of the tests with the risk of the subject developing cancer.

[0059] A diagnostic kit of the present invention may include reagentsfor performing functional determinations of cytotoxicity of diagnosticcells (i.e., reagents for performing tests measuring cytokine releasefrom primed and activated diagnostic cells, and/or reagents forperforming tests of tumor cell killing by primed and activated cells).Accordingly, a diagnostic kit of the present invention may include atleast one container sufficiently sized to include at least one primingagent, at least one activating agent, and at least one reagent fordetermining a functional measure of cytotoxicity of diagnostic cells ofa subject. If the functional measure of cytotoxicity is the percent oftumor cells killed by the primed and activated diagnostic cells of thesubject, then the kit may contain a sample of target cancer cells (e.g.,tumor cells) that the primed and activated diagnostic cells of thesubject may be tested against. Diagnostic kits for determiningfunctional measures of cytotoxicity of diagnostic cells will includeprinted instructions for conducting the appropriate tests, and mayinclude instructions for isolating diagnostic cells from a subject, andinstructions for conducting the tests themselves. The printedinstructions will also include instructions for correlating the resultsof the tests with the risk of the subject developing cancer.

[0060] Kits useful for the prevention of cancer according to thepreventative methods of the present invention will include at least onecontainer sufficiently sized to include at least one reagent forincreasing A₁ adenosine receptor expression in cells (i.e., macrophages,monocytes, peripheral blood stem cells, promonocytes) of an at-riskpatient, and/or at least one reagent for increasing binding of A₁adenosine receptor ligands to these cells, and/or at least one reagentfor directly priming and/or activating cells of the subject in vitro orin vivo. A kit useful for prevention of cancer according to thepreventative methods of the present invention may alternatively oradditionally include at least one sample (or dose) of macrophages,monocytes, promonocytes or peripheral blood stem cells cloned or treatedwith, for example, cDNA encoding A₁ adenosine receptors, drugs orischemia in order to increase A₁ adenosine receptor expression, functionand tumoricidal effect. Prevention kits of the present invention willinclude printed instructions for administering to at-risk subjects thereagent or reagents useful in preventing cancer.

[0061] The following Examples are provided to illustrate the presentinvention, and should not be construed as limiting thereof.

EXAMPLE 1 Preparation of Mouse Macrophage Cell Culture

[0062] Endotoxin-sensitive (LPS-sensitive) mouse macrophages (ATCCIC-21) are grown to confluency in RPMI medium containing 2 mM glutamine,1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodiumpyruvate and 10% fetal bovine serum in 24-well culture plates or 75 mm²culture flasks in a humidified CO₂ incubator.

[0063] Endotoxin-resistant (LPS-resistant) mouse macrophages (ATCCWB264-9C) are grown to confluency in minimum essential medium (Eagle)with Earle's balanced salt solution containing 10% fetal bovine serum in24-well culture plates or 75 mm² culture flasks in a humidified CO₂incubator.

EXAMPLE 2 In vitro Cytotoxicity Experiments

[0064] IC-21 macrophages, WBC264-9C macrophages, and WBC264-9Cmacrophages previously treated with cisplatin (2.5 μM for 24 hours) areincubated with serum free media for 24 hours before the experiment.

[0065] B-16 melanoma cells are washed with serum free RPMI medium bycentrifugation. 25-30 μCi of Na ⁵¹Cr is diluted to 1 ml of RPMI andtransferred to the melanoma cells suspension. The melanoma cells areincubated with Na ⁵¹Cr at 37° C. for 1 hour. The unincorporatedradioactivity is removed by centrifugation at 1000×g for 10 minutes. Themelanoma cells are allowed to leak for 2 hours and then washed again.The final suspension is diluted in RPMI. The cell number is counted in ahemocytometer.

[0066] Macrophages are washed with medium containing 100 mM MgCl₂, 2U/ml adenosine deaminase and 10% fetal bovine serum. Macrophages arethen challenged with 1 μM PMA in the above medium for 2 hours and thenstimulated with different concentrations of (0-1 μM) of2-chloro-N⁶-cyclopentyl adenosine (CCPA) for 30 minutes. The macrophagesare then incubated with ⁵¹Cr-labelled B-16 melanoma cells at a ratio of20:1. Six hours later, a fraction of the supernatant is aspirated andcounted in a gamma counter.

[0067] Spontaneous release of the radioactivity is measured from thewells that do not contain macrophages. Total releasable counts aremeasured by lysing the melanoma cells in hypotonic medium.

[0068] The results are expressed as percentage (%) ⁵¹Cr release asfollows:${{percentage}\quad (\%)^{51}{Cr}\quad {release}} = \frac{{{Experimental}\quad {release}} - {{spontaneous}\quad {release}}}{{{Total}\quad {releasable}\quad {counts}} - {{spontaneous}\quad {release}}}$

[0069] Percentage ⁵¹ Cr release is a measure of the lysing of cells(i.e., killing of cells), and is therefore an indicia of cytotoxicity.The results of this experiment are illustrated in FIG. 1, in whichpercentage ⁵¹Cr release is expressed as a function of increasing logmolar CCPA, and in which filled-in squares represent IC-21 macrophagedata and open squares represent WBC264-9C macrophage data.

[0070] This same protocol is repeated, except that P815 mousemastocytoma tumor cells are used instead of the B-16 melanoma cells. Theresults of this experiment are shown in FIG. 2. As illustrated by theresults of these experiments, primed and activated macrophages fromLPS-sensitive animals exhibit a significantly higher cytotoxicityagainst tumor cells when compared to the cytotoxicity of primed andactivated macrophages from LPS-resistant animals.

[0071] The protocol is also repeated using cultured human macrophages(SC cells) and A375 human melanoma cells. The results of this experimentare shown in FIG. 3.

EXAMPLE 3 Preparation of Mouse Macrophage Membranes

[0072] The macrophages described in Example 1 are lysed in 10 mM TrisHCl buffer, pH 7.4, containing 5 mM EDTA, 10 μg/ml soybean trypsininhibitor, 10 μg/ml benzamidine and 2 μg/ml pepstatin. The macrophagesare homogenized using a cell sonicator to produce a homogenate. Thehomogenate is centrifuged at 1000×g for 10 minutes. The supernatant iscentrifuged again at 40,000×g for 20-30 minutes at 4° C. The membranepellet is resuspended in 50 mM Tris-HCl buffer, pH 7.4, 10 mM MgCl₂, 1mM EDTA, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamidine, 2μg/ml pepstatin and 5 U/ml adenosine deaminase.

EXAMPLE 4 Saturation Binding Experiments

[0073] Saturation binding experiments are performed in membranefractions (10-20 μg protein) from IC-21 macrophages, WBC264-9Cmacrophages, and WBC264-9C macrophages previously treated with cisplatin(2.5 μM for 24 hours). Incubation of the macrophage fractions with theA₁ adenosine receptor ligand ¹²⁵I-BWA844U (see Patel et al., J.Pharmacol. Exp. Ther. 33:585 (1988) in concentrations ranging from1.25-20 nM was carried out at 37° C. for 1 hour. Non-specific binding isdetermined in the presence of 1 mM theophylline. Following incubation,membrane bound radioactivity is separated by filtration over a cellharvester and counted in a gamma counter. The data is analyzed bynon-linear regression using GraphPad Prism software. Thc B_(max) andk_(d) values are calculated from the analyzed data.

[0074] Results of this experiment are shown in FIG. 4, in which B_(max)(expressed in units of fmol/mg protein) is plotted as a function ofincreasing concentration ¹²⁵I-BWA844U. Solid squares represent data fromIC-21 macrophage membranes, while solid triangles represent data fromWBC264-9C macrophage membranes. These results illustrate macrophagemembranes from LPS-sensitive mice bind more A₁ adenosine receptor ligandthan do macrophage membranes from LPS-resistant mice.

[0075] This same protocol is repeated using membranes from humanmacrophages (SC cells). The solid squares represent data from membranesnot treated with cisplatin, while solid triangles represent data frommembranes treated with cisplatin (2.5 μM for 24 hours). The results ofthis experiment are shown in FIG. 5. These results show thatpre-treating macrophage membranes with cisplatin increased the B_(max)of the macrophage for the A₁ adenosine receptor ligand by approximatelythree-fold.

EXAMPLE 5 Competition Experiments with Human Recombinant MCP Protein

[0076] Competition experiments are performed with differentconcentrations of human recombinant MCP-1 protein with a fixedconcentration (0.1-0.2 nM) of ¹²⁵I-BWA844U in macrophage membranes fromIC-21 macrophages, WBC264-9C macrophages, and WBC264-9C macrophagespreviously treated with cisplatin (2.5 μM for 24 hours). Non-specificbinding is determined in presence of 1 mM theophylline. Membrane boundradioactivity is separated by filtration over a cell harvester. The datais analyzed by non-linear regression analysis using GraphPad prismsoftware and the K_(i) value is calculated.

[0077] Affinity for MCP-1 is lower (i.e., the K_(i) is higher) inmembranes from WBC264-9C macrophages than it is for membranes from IC-21macrophages. Pretreatment with cisplatin increases the affinity ofmembranes from WBC264-9C macrophages for MCP-1.

EXAMPLE 6 Functional Experiments: TNF-α Release

[0078] IC-21 macrophages, WBC264-9C macrophages, and WBC264-9Cmacrophages pretreated with cisplatin (2.5 μM for 24 hours) are washedwith medium containing 100 mM MgCl₂, 2 U/ml adenosine deaminase and 10%fetal bovine serum. Macrophages are then challenged with 1 μM PMA in theabove medium for 2 hours and then stimulated with differentconcentrations of (0-1 μM) of 2-chloro-N⁶-cyclopentyl adenosine (CCPA)at 37° C. for 1-6 hours. The supernatant is collected and the TNF-αlevels are estimated by commercially available ELISA kits (R & DSystems, Minneapolis, Minn.).

[0079] Following priming with PMA and activation with CCPA, TNF-αrelease is lower in WBC264-9C macrophages than it is in IC-21macrophages. Pretreatment of the WBC264-9C macrophages with cisplatinincreases TNF-α release from WBC264-9C macrophages.

EXAMPLE 7 In vivo Experiments for Prevention of B-16 Mouse Melanomaswith LPS-Sensitive and LPS-Resistant Macrophages

[0080] Mice, tumors: Mice are implanted subcutaneously by injection with1×10⁵ mouse B-16 melanoma cells in the flank. Animals are sorted intothe various treatment groups (see Table 1), and treatments are initiated30 minutes prior to injections of B-16 melanoma tumor cells. Estimatedtumor weight is calculated using the formula:${{Tumor}\quad {Weight}\quad ({mg})} = \frac{w^{2} \times l}{2}$

[0081] Where w width and l=length in mm of B-16 melanoma

[0082] Treatment: The treatment plan for animals in this study is shownin Table 1, below. Mice are sorted on Day 1 into nine groups with tenanimals per group. Dosing via intravenous (TV) tail vein injection orintraperitoneal (IP) is initiated on Day 1 for all agents. Mice aredosed with IV treatments 4 times a week for 4 weeks (qd×4×4 wk). Micearc dosed with IP treatments once a day for 5 days (qd×5). Tumor calipermeasurements are obtained twice weekly for all mice. Animals are weighedtwice weekly. The study is terminated on Day 60.

[0083] Endpoint: The tumor growth delay (TGD) method is used in theseexperiments. In the TGD method, each animal is euthanized when its B-16melanoma reaches a size of 2.0 g; this is considered a cancer death.Mean Day of Survival (MDS) values are calculated for all groups.Treatment-effected mean increases in survival of various groups arecompared to each other and to the mean survival times of controltumor-bearing mice (no treatment) and tumor-bearing mice receiving onlyvehicle (PBS) or tumor bearing mice receiving only naïve macrophages.${{Time}\quad {to}\quad {endpoint}\quad ({calculated})} = {{{Time}\quad {to}\quad {exceed}\quad {endpoint}\quad ({observed})} - \frac{{Wt}_{2} - {{endpoint}\quad {weight}}}{\frac{{Wt}_{2} - {Wt}_{1}}{D_{2} - D_{1}}}}$

[0084] where:

[0085] Time to exceed endpoint (observed)=number of days it takes foreach tumor to grow past the endpoint (cut off) size. This is the day theanimal is euthanized as a cancer death.

[0086] D₂=day animal is euthanized.

[0087] D₁=last day of caliper measurement before tumor reaches theendpoint.

[0088] Wt₂=tumor weight (mg) on D₂

[0089] Wt₁=tumor weight (mg) on D₁

[0090] Endpoint weight=predetermined “cut-off” tumor size for the modelbeing used.

[0091] As shown in Table 1, below, treatment according to the aboveprotocol causes either complete tumor prevention or partial tumorprevention in an animal. Also, therapy may prevent a neoplasm's growthto a small size but not reach the 2.0 g cut-off. Specifically, there iscomplete or partial prevention of tumor growth in animals pretreatedwith primed and activated LPS-sensitive macrophages (IC-21). There isless tumor growth prevention in animals pretreated with primed andactivated LPS-resistant macrophages (WBC264-9C) than in the animalspretreated with primed and activated LPS-sensitive macrophages (IC-21).Tumor growth prevention in animals pretreated with primed and activatedLPS-resistant macrophages pretreated in vitro with cisplatin (2.5 μM for24 hours) (WBC264-9C+cisplatin) is greater than that observed in animalspretreated with primed and activated LPS-resistant macrophages(WBC264-9C) without cisplatin in vitro pretreatment or cisplatinadministered intraperitoneally alone. There is complete or partial tumorprevention in animals treated with naïve (without priming or activation)LPS-resistant macrophages pretreated in vitro with cisplatin (2.5 μM for24 hours) (WBC264-9C+cisplatin).

[0092] Toxicity: Animals are weighed twice weekly during the study. Miceare examined several times a week for clinical signs of drug-relatedside effects.

[0093] Statistics: The unpaired t-test and Mann-Whitney test (analyzingmeans and medians, respectively) are used to determine the statisticalsignificance of any difference in survival times between a treatmentgroup and the control group. All statistical analyses are conducted at plevel of 0.05 (two-tailed). P-values obtained dictate the percent chancethat random sampling from identical populations would lead to adifference larger than observed (p=0.03=3% chance). Prism (GraphPad)version 3a is used for all statistical analysis and graphs. TABLE 1Treatment Groups Group No. n Drug/Agent Dose Route Schedule 1 10 Notreatment 2 10 PBS 0.2 ml IV qd × 4 × wk 3 10 Naïve Macro/IC-21 1 × 10⁷IV qd × 4 × 4 wk 4 10 Naïve Macro/WBC264-9C 1 × 10⁷ IV qd × 4 × 4 wk 510 Naïve Macro/WBC264-9C + 1 × 10⁷ IV qd × 4 × 4 wk Cisplatin 1 × 10⁷ IVqd × 4 × 4 wk 6 10 P/AMacro/IC-21 1 × 10⁷ IV qd × 4 × 4 wk 7 10P/AMacro/WBC264-9C 1 × 10⁷ IV qd × 4 × 4 wk 8 10 P/AMacro/WBC264-9C + 1× 10⁷ IV qd × 4 × 4 wk Cisplatin 9 10 Cisplatin 100 mg/m² IP qd × 5#macrophages are not treated with PMA and CCPA. Macrophages are injectedintravenously (IV) in 0.2 ml PBS.

[0094] The foregoing is illustrative of the present invention and is notto be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of determining a subject's riskfor developing cancer, comprising: obtaining a sample of diagnosticcells from a subject, said diagnostic cells selected from the groupconsisting of macrophages, monocytes, promonocytes and peripheral bloodcells; and then determining a measure of cytotoxicity of the diagnosticcells for target cancer cells, the measure of cytotoxicity correlatingnegatively with the risk for developing cancer; whereby said subject'srisk for developing cancer is determined.
 2. The method according toclaim 1, wherein the measure of cytotoxicity is determined by evaluatingthe affinity of the diagnostic cells for at least one A₁ adenosinereceptor ligand.
 3. The method according to claim 1, wherein the measureof cytotoxicity is determined by evaluating the number of A₁ adenosinereceptors on the diagnostic cells.
 4. The method according to claim 1,wherein the measure of cytotoxicity is determined by evaluating theaffinity of the diagnostic cells for MCP-1 protein.
 5. The methodaccording to claim 1, further comprising the steps of: priming thediagnostic cells by contacting the diagnostic cells with a priming agentin an amount sufficient to prime the diagnostic cells; and activatingthe diagnostic cells by contacting the diagnostic cells with anactivating agent in an amount sufficient to induce cytotoxicity in thediagnostic cells; wherein the priming and activating steps occur priorto determining the measure of cytotoxicity of the diagnostic cells fortarget cancer cells.
 6. The method according to claim 5, wherein themeasure of cytotoxicity is determined by evaluating the release ofcytotoxins from the diagnostic cells.
 7. The method according to claim5, wherein the cytotoxin is tumor necrosis factor α (TNF-α.
 8. Themethod according to claim 5, wherein the measure of cytotoxicity isdetermined by evaluating the percentage of target cancer cells killed bythe diagnostic cells.
 9. The method according to claim 5, wherein theactivating agent is an A₁ adenosine receptor agonist.
 10. The methodaccording to claim 5, wherein the activating agent is conjugated to alipid.
 11. The method according to claim 5, wherein said priming agentis selected from the group consisting of phorbol myristoyl acetate(PMA), lipopolysaccharide (LPS), interferon gamma (IFNτ),granulocyte-macrophage colony stimulating factor (GMCSF), andf-met-leu-phe (fMLP).
 12. The method according to claim 5, wherein saidpriming agent is conjugated to a lipid.
 13. The method according toclaim 1, wherein said subject is human.
 14. The method according toclaim 5, wherein said measure of cytotoxicity is determined byevaluating the affinity of the diagnostic cells for at least one A₁adenosine receptor ligand and by evaluating the percentage of targetcancer cells killed by the diagnostic cells.